It is known that, in living organisms and life phenomena, free radical species such as nitrogen monoxide act as a second messenger for signal transduction, and they exert various physiological functions, for example, control of blood pressure in the circulatory system and the like. Reactive oxygen, which is one type of free radical species, is a generic term for superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen and the like. Among them, superoxide anion and hydrogen peroxide have already been revealed to exert important physiological functions in the immune system and the like. There are also many articles reporting that hydroxyl radical has been found to be involved in vascular disorders or brain disorders after ischemia or DNA modification by ultraviolet, and the radical is considered to be a reactive oxygen species having particularly high harmful nature in relation to causes and pathologies of diseases. As for singlet oxygen, its role and the like has little been revealed so far. Recently, some evidence has been obtained suggesting that singlet oxygen is a reactive species in the photodynamic therapy, which is one of cancer therapies, and is generated by various kinds of oxidases, peroxidases and the like in living organisms, which suggests its role of important physiological functions.
Elucidation of the role of reactive oxygen species in living organisms has thus become more and more important. However, there are many problems in methods for measurement of the species. As for methods for measurement of hydroxyl radical, various reports have been made on its measurement by the electron spin resonance (ESR) method. However, the ESR method has fundamental difficulty of using living cells as measurement samples, and the measurement and the evaluation at an individual cell level are practically impossible. A method is also known in which DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate, Molecular Probes, catalog No. D-399), which is capable of measuring wide variety of reactive oxygen species, is used together with an inhibitor against generation of other reactive oxygen species, and hydroxyl radical is detected under a microscope. However, results obtained in the coexistence of the inhibitor includes some factors different from reactions in a living organism. In addition, DCFH-DA is very susceptible to autoxidation, and for this reason, background fluorescence by autoxidation interferes the detection when the same field is needed to be observed several times. The method is also very inconvenient from viewpoint of operability and storability as it requires operations in the dark.
Ten or more kinds of methods have been known as methods for measurement of singlet oxygen, including the chemiluminescence method, the ESR method, and the luminescence method. However, these methods in common give only low specificity and sensitivity, and thus they are not reliable methods (as for the method for specific detection of singlet oxygen, see, Nagano, T., et al., Free radicals in Clinical Medicine, Vol.7, pp.35–41, 1993 and the like). The DCFH-DA may be used in the measurement of singlet oxygen, however, problems of DCFH-DA, per se, cannot be avoided. Therefore, it has been desired to develop a method for measurement of singlet oxygen species, which can be used in research of reactive oxygen species and is excellent in specificity and sensitivity with easy operability.